RNA-seq Library Preparation from HEK293T Cells

Nuclear RNA was prepared from HEK293T (kidney) cells. Briefly, cells were lysed on ice for 5 min in 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 0.05% NP-40, and nuclei were spun at 2,500g for 3 min and then resuspended in QIAzol for RNA isolation using the miRNeasy kit according to the manufacturer’s instructions (Qiagen). The RNA-seq library was created using the Illumina TruSeq RNA Sample Preparation Kit v2 with the standard protocol, and sequenced on one lane of the HiSeq 2000 platform (100 bp, paired-end). Data are available at NCBI as accession number SRP041943. The database of annotated protein coding and noncoding genes (41,409 genes and 171,904 transcripts in total) was produced by merging all annotated genes from the RefSeq database^{29 (link)}, the UCSC Browser^{24 (link)} and the Ensembl database^{30 (link)}.